Hi fellow friends...
I'm interested in identify and isolating polyhydroxy alkaloid (imino sugars) from my sample (plant). I've gone through many journal and most of it use ion exchange resins in isolate and purifying the imino sugars. I do have Amberlite IR-120 (H+ form), Dowex 1x2 (OH- from) and Amberlite CG-50 (NH4+ form) but I'm lack with an experience and knowledge regarding the IEC.
Here, I use paper of Kato et al. (2003) as my reference.
1. A 50% MeOH extract of seeds (850 g) of C. australe
was applied to a column of Amberlite IR-120B (500
mL, H+ form).
My question : What does it mean by 500 mL? Is it the volume of resin is 500 mL or 500 mL volume of 50% MeOH extract of the seeds? If the 500 mL is the volume of the extract, how much of resin should I use? If I used a sample with 200 g, does it mean the 500 mL become 118 mL (using ratio calculation)?
2.The 0.5 M NH4OH eluate was concentrated to give a brown oil (35.6 g), which was chromatographed over a Dowex 1-X2 column (3.8×90 cm,
OH− form) with H2O as eluant (fraction size 15 mL)?
My question: If my sample after elution is less than 35.6g, should I use another dimension of column? How do we consider it? What is the meaning of fraction size 15 mL??
3. The H2O eluate was divided into two pools: A (fractions 16–50, 20.6 g) and B (fractions 51–86, 3.78 g).
My question: Does it means during elution, I need to collect my sample little by little and check their purity?
I hope there is someone that can guide me in detailing this IEX method. I appreciate your help and thank you in advance.
I will answer the questions using the numbers provided above.
Dear Jack...thank you for your help. I'm finally can understand what is the meaning of the methods. But can you do me a favor? Could you elaborate more on point number one? What media do you mean in the 1st answer? Is it the buffer or the extract? And how many gram resin should I use? Again, thank you an advance Jack. =)
Not knowing the bulk density of the resin, I can't tell how many grams is equal to 500 mL of resin. The best way to determine that is to fill a tared 500 mL beaker to the 500 mL mark and determine the mass. By "media", I meant the chromatographic media, the Amberlite IR-120B. Note that they specified the H+ form of the resin- this is important, because the desired compound(s) may not bind if the resin has other counter ions..
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